The activity of the enzyme responsible for the synthesis of cyclic AMP in Escherichia coli is regulated in a complex manner that is not completely understood. Major protein factors involved in this regulation are components of the sugar transport system known as the phosphoenolpyruvate:sugar phosphotransferase system (PTS). Consequently, considerable attention has been focused on protein components of the PTS. Nuclear magnetic resonance spectroscopy (NMR) has been used to elucidate the solution structure of the aminoterminal domain of enzyme I (EIN) of the PTS. The data show that the structure in solution is essentially identical to that in the recently elucidated X-ray crystal structure. NMR has also been used to characterize the interaction of EIN with the histidine-containing phosphocarrier protein HPr of the PTS. The binding surface on EIN is primarily located in the alpha-domain. A model of the EIN/HPr complex was proposed in which helix 1 and the helical loop of HPr slip between the two pairs of helices constituting the alpha-domain of EIN. A functional role for the kink between helices H2 and H2' is proposed where it provides a flexible joint for the interaction to take place. Specificity studies of full-length enzyme I compared to the aminoterminal domain of enzyme I established that E. coli enzyme I will effect phosphoryl transfer specifically to HPr from E. coli. However, the aminoterminal domain of enzyme I shows a relaxed specificity and will effect phosphotransfer to HPrs from several bacterial species. These studies demonstrate that the carboxyl-terminal domain of enzyme I confers on the protein the capability to accept a phosphoryl group from phosphoenolpyruvate as well as a discriminator function that allows the intact protein to promote effective phosphoryl transfer only to E. coli HPr. Characterization of the region of the genome of Mycoplasma capricolum upstream of the portion encompassing the genes for enzymes I and IIAglc of the PTS showed that it contained a gene cluster encoding a family of genes responsible for the metabolim of pyruvate. The gene encoding enzyme IIAglc of the PTS of M. capricolum was expressed, purified and the protein characterized. It has a higher pI than previously characterized enzymes IIAglc and is unreactive against antibodies directed against the corresponding proteins from Gram-positive or Gram-negative bacteria.